Allogeneic hematopoietic stem cell transplantation (HSCT) is curative for many patients with malignant diseases. However, many patients lack HLA-matched donors, particularly those of minority or mixed race or ethnicity. Almost all patients have available haploidentical family donors, the use of which would significantly increase the availability of HSCT as a treatment modality. However haploidentical HSCT results in an increased frequency and severity of acute graft versus host disease (aGvHD), mediated by alloreactive donor T cells. Although non-selective T cell depletion of haploidentical donor grafts effectively prevents severe aGvHD, it delays immune reconstitution and increases both infectious complications and relapse rates limiting the success of haploidentical HSCT. Several strategies have therefore been developed to selectively remove or inactivate alloreactive T cells within the donor T cell pool to create a cellular product capable of conferring beneficial immune reconstitution without severe aGvHD. One such strategy is induction of alloantigen-specific hyporesponsiveness in donor T cells by recipient alloantigen presentation with concurrent co-stimulatory blockade ("alloanergization"). This strategy was successfully employed in 2 pilot studies where large doses of haploidentical alloanergized donor T cells were infused en mass with donor bone marrow (BM), resulting in less severe aGvHD than seen historically in recipients of unmanipulated haploidentical BMT. No deaths were attributable to CMV or other viral infections, suggesting donor T cells retained functional pathogen-specific immunity, and only 1 of 12 evaluable patients developed chronic GvHD. Eight of 24 high risk patients survive disease-free with normal performance scores (median follow-up 8 years). Although, these studies demonstrate that favorable long-term functional outcome can occur after haploidentical alloanergized BMT, two important questions remained unanswered. Firstly, what is the optimal dose of alloanergized T cells, and secondly, is antigen-specific immune reconstitution improved by infusing such cells? We propose a new study to answer these questions. An adaptive, dose escalation design and more tolerable conditioning regimens will be used. Larger doses of haploidentical donor stem cells from CD34-selected peripheral blood will be given followed by delayed infusion of haploidentical donor T cells alloanergized with an improved method. In Specific Aim 1, we will determine the feasibility of multi-institutional conduct of the novel cell-processing and Phase I clinical trial. Specific Aim 2 focuses on measurement of pathogen- and tumor-associated antigen-specific T cell immunity in samples from study patients. Specific Aim 3 will quantify and characterize the function of donor-derived CD4+ T regulatory cells in patients so treated, thus further examining a novel mechanism by which anergization strategies appear to contribute to in vivo alloantigen hyporesponsiveness. The data derived from the proposed experiments will provide the necessary clinical and scientific information to design further pivotal studies that will determine the potential of this novel approach to ameliorate HSCT morbidity and mortality. PUBLIC HEALTH RELEVANCE: Many patients with diseases potentially curable by hematopoietic stem cell transplantation cannot find matched donors and although most have "mismatched" family donors, use of mismatched family donors often results in transplants complicated either by the donor immune cells attacking the patient (so-called Graft versus Host Disease (GvHD)) or by poor immune function if the donor immune cells are removed. We have shown that mismatched family donor immune cells "tolerized" to the transplant recipient may reduce GvHD after mismatched transplantation, but we do not know the best number of tolerized immune cells to administer or what immune benefits they may produce. Here, we will conduct a clinical trial to determine the number of tolerized donor immune cells necessary to improve immune function after mismatched HSCT without contributing to GVHD, and perform laboratory experiments to characterize the mechanisms by which these goals are achieved